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Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2002
EXPERIMENTS
Preparation of ODNs and ODN-modified nanospheres.
All ODNs used in this
study were prepared on a fully automated DNA synthesizer (Oligo 1000M DNA
synthesizer, Beckman). Purification, storage, and quantification were carried out
following previously described methods (T. Ihara, Y. Maruo, S. Takenaka, M.
Takagi, Nucleic Acids Res., 1996, 24, 4273.). The spheres (FluoSphere, Molecular
Probes, Inc., Eugene, OR) used in this study have carboxylic groups on their surface
(1 carboxylate/9 Å2) (R. P. Haugland, Handbook of fluorescent probes and research
chemicals, Edn. 7; Molecular Probes, Inc., OR, 1999.). Amino-terminated
oligonucleotides, 5cWT15 or 3cWT15 were anchored onto the spheres using
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) in NaHCO3/Na2CO3
buffer solution (pH 9.8). As a typical procedure, 300 µL of aqueous buffer solution
(0.2 M NaHCO3 /Na2CO3 ) containing 120 nmol ODNs (5cWT15 or 3cWT15), 1.1
x 1014 spheres (carrying 12 µmol of carboxylic groups), and 12 µmol EDAC was
shaken for 12 h at room temperature. After the capping of residual activated surface
carboxylic groups with 2 mg glycine, the ODN-modified nanospheres were isolated
from the free 5cWT15 or 3cWT15 and low-molecular weight by-products by GPC
(Sephadex G-200). The unreacted free 5cWT15 and 3cWT15 were quantified using
a UV-vis spectrophotometer (Hitachi U-3010) to estimate the surface coverage of
the spheres.
Fluorescence microscopy.
A fluorescence microscope (Olympus BX51) equipped
with a CCD camera was used for observing the aggregates. For selective
observation of each sphere, appropriate sets of optical filters were used; the filters
sets with the band pass of 470-490 nm for excitation and 515-550 nm for emission,
and that of 520-550 nm for excitation and > 580 nm for emission were used to
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observe the yellow-green and the red spheres, respectively. One µL of the sample
solutions containing 5.7 x 108 of both spheres (carrying 210 fmol of 5cWT15 and
3cWT15, respectively) and 40 mM Hepes (pH 7.0) were used for the observation.
The solutions of the aggregates also contained 10 mM MgCl2 and 210 fmol of
WT45AGG or MT45AGT. After 10 minutes of making the solutions, these were put
between slide and cover glasses, and then subjected to microscope inspection at
room temperature. The two photos taken through each of the filter sets were
superposed over each other using image processing software (Adobe Photoshop 3.0,
Adobe Systems).
Observation of the FRET between the spheres.
The emission spectra were
measured using a fluorescence spectrophotometer (Hitachi F-2500) with an optical
filter to screen (< 500 nm) the intense Rayleigh scattering. The sample solutions
used here were the diluted solutions of those used for the fluorescence microscopy
for ca. 500 times with the aqueous buffer (40 mM Hepes (pH 7.0), 10 mM MgCl 2)
to make 1 mL solutions, which contained 1.0 x 109 of both spheres and, in the
aggregates solutions, 400 fmol of WT45AGG or MT45AGT as well. All spectra were
obtained by excitation at 470 nm at room temperature just after dilution. The spectra
were corrected for the absorption of the sample solution themselves and the optical
filter, and the characteristics of the photomultiplier.
SUPPLIMENTARY RESULTS
TEM observation of the spheres.
The spheres before and after the ODN
modification were observed by transmission electron microscopy (TEM). The
spheres were stained with uranyl acetate, which should strongly bind with
phosphates in the backbone of the ODNs. The circular rims of the particles
appeared to blacken with ODN modification, indicating that the ODNs were
anchored onto the spheres and the modification reaction did not cause significant
influence on the sphere body itself.
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before modification
after modification
Preliminary study on hybridization.
5cWT15 was immobilized onto the spheres
with 1 m diameter. These spheres precipitate easily by centrifuge. Two kinds of
BODIPY-ODN conjugates were synthesized by ordinary coupling method via
activated ester of BODIPY. One of them is the 15mer complementary to 5cWT15
(corresponds to 5’-end 15mer of WT45AGG), other is the 15mer carrying one base
mutation (corresponds to 5’-end 15mer of MT45AGT). Hybridization experiments
were carried out for 0.1 M conjugates and immobilized 5cWT15 in
CH3
N
F
B
CH2 CH2
N
F
O
O
O
C N (CH2)5 C O N
H
CH3
+
H 2N
O
H3CO
O
O
P
O O
NNN...NN
CH3
N
F
B
O
O
CH2 CH2 C N (CH2)5 C N
H
H
N
F
O
O
P
O O
NNN...NN
CH3
BODIPY-ODN
H3CO
Structure and synthesis scheme
the buffer solution containing 10 mM MgCl2 and 40 mM Hepes (pH 7). After
incubation for 3 h at 25°C, the spheres were precipitated from the dispersed
solutions by centrifugation (15,000 rpm for 1 min). Then, the concentrations of free
BODIPY-ODN conjugate in supernatants were measured using its fluorescence. The
hybridization efficiency of complementary and mutant conjugate, the rate of
capturing conjugates, were 72 % and 5 %, respectively. Almost of all captured
complementary BODIPY-ODN were recovered from the spheres by the 3 M urea
treatment. It means that hybridization takes place on the ODN-modified spheres and
fidelity in the recognition is retained in this heterogeneous system.
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Sequence specific hybridization on the ODN-modified spheres
The spheres carrying short ODNs that have complementary 10mer sequence
without spacer such as dT10 was prepared to check the requirements for ODNs that
were immobilized onto the spheres. The 10mer-immobilized spheres scarcely
captured complementary BODIPY-ODN conjugate. This result indicates that the
spacer and/or longer sequence was required for effective hybridization on the
spheres.
Requirements for ODNs immobilizd onto the spheres
Dependence of the sphere aggregations on the salt concentration.
The
concentration of salt is one of the critical parameters for the studies of fluorescence
microscopy and FRET. The concentration was decided according to the results of
salt titrations.
Into the mixed dispersed solutions of the both spheres, concentrated aqueous of
MgCl2 was titrated with monitoring the turbidity at 400 nm at 25 °C. The initial
solutions contains 5.7 x 1011 of both of the spheres and 210 nmol of WT45AGG or
MT45AGT in 1 mL buffer solutions (40 mM Hepes (pH 7)).
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Although the both solutions became turbid with increasing the concentration of
Mg2+, the turbidity of the solution containing the target, WT45AGG, increased from
less Mg2+ concentration than that containing the mutant, MT45AGT. The Mg2+
concentration that could give the difference in the extent of aggregation between
the both solutions ([MgCl2] = 10 mM) was adopted as one of the conditions in
microscopy and FRET studies.
*Much difference in the ionic strength in the two systems (dT20-modified sphere and
sphere R-G systems) is not surprising, because divalent cations such as Mg2+ have
extremely powerful effect compared with monovalent cations such as Na + on
duplex formation of nucleic acids (i.e., M. T. Record, C. F. Anderson Jr., T. M.
Lohman, Quart. Rev. Biophys. 1978, 11, 103; G. S. Manning, Quart. Rev. Biophys.
1978, 11, 179.).
0.35
Absorbance at 400 nm
0.30
0.25
with WT45
0.20
AGG
0.15
with MT45
AGT
0.10
0.05
0.00
0
5
10
15
20
[MgCl 2] / mM
Dependence of DNA-directed sphere aggregation
on MgCl2 concentration
Aggregation of dT20-modified spheres.
These spheres showed aggregation in the
presence of complementary RNA homopolymer, polyA under the appropriate
conditions (1.0 M NaCl, 40 mM Hepes (pH 7.0)) (b). On the other hand, the spheres
were fully dispersed in the presence of polyC and polyU under the same conditions
(a). In addition, the aggregation with polyA was entirely suppressed by the addition
of 5 M urea. The aggregation was reversible for the change of salt concentration and
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temperature; this is the feature of hybridization of nucleic acids.
a
b
10m
10m
Preliminary aggregation experiment using RNA homopolymer.
FRET imaging.
Using appropriate set of optical filters, we could observe the image
of FRET. Bright red image was observed for well networked yellow aggregates with
WT45AGG. On the other hand, the solution containing the aggregates with
MT45AGT gave just slightly emitted points. As mentioned in the manuscript,
effective FRET takes place only between the two spheres neighboring each other.
These data could be a direct evidence indicating the mutual connection of the
experimental data of Figure 2 and Figure 3.
* It is likely that the aggregation in the presence of MT45AGT mostly due to the
hydrophobic nonspecific interaction between the spheres in the presence of MgCl 2.
Subtle difference in the propensity for aggregation of sphere R from that of sphere
G (sensitivity for ionic strength) seems to make the spheres gathered separately;
each of the spheres might aggregate sequentially. Prevention of this nonspecific
interaction would contribute improvement of the sensitivity of this method.
Increasing the number of modified DNAs or hydrophilic chemical modification of
sphere surface should help to improve the sensitivity of the present method. Such
extensions are in progress.
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FRET imaging of the sphere aggregates